In type 1 diabetes (T1D) research there remains a critical need for pursuing a systems level understanding of early stage autoimmune-mediated molecular mechanisms which trigger the specific destruction of pancreatic ? cells. Thus, the overall objectives of this application are to discover novel in vivo signaling pathways and regulatory networks that contribute to early stage ? cell stress and death, and to identify potential therapeutic targets for intervention and early diagnostic biomarkers. These objectives will be pursued by applying enabling proteomics technologies focusing on posttranslational protein modifications (PTMs) to unique sets of human islet samples. Specifically, we hypothesize that posttranslational regulation, involving phosphorylation, S- nitrosylation, and S-glutathionylation, represents a fundamental triggering mechanism of ? cell dysfunction preceding overt T1D. To address the limitations associated with clinical samples for studying dynamic signaling networks, our plan utilizes three complementary model systems: a) human islets isolated by laser-capture microdissection from clinical human pancreatic tissues, b) human islets treated with cytokines in vitro, and c) human islets transplanted in 'humanized' mice for recapitulating in vivo islet dysfunction. Studies in Aim 1 will identify signaling pathway and networks involved in early stage ? cell dysfunction and apoptosis using global PTM focused proteomic technologies. In Aim 2, we will apply a targeted quantification approach to verify specific regulatory networks and PTMs of interest using samples from individual patients as well as time- course islet samples from 'humanized' mice. In Aim 3, we will evaluate protein targets discovered in the first two aims, determining their potential as ? cell specific markers for early T1D diagnosis in serum, and their potential functional roles in ? cell apoptosis through additional cultured islet studies. Together, we anticipate that this project will demonstrate a new paradigm of systems level study of posttranslational regulation of ? cell dysfunction and apoptosis, and will provide a novel integrative view of the early stage regulatory networks that potentially trigger ? cell apoptosis and T1D. This work will also provide a rich molecular data resource for the consortium on beta-cell death and survival (CBDS), the human islet research network (HIRN), and the diabetes community in generating new hypotheses for functional studies.